Age-Related Inflammation and Oxidative Stress in the Cochlea Are Exacerbated by Long-Term, Short-Duration Noise Stimulation.

We have previously reported that young adult rats exposed to daily, short-duration noise for extended time periods, develop accelerated presbycusis starting at 6 months of age. Auditory aging is associated with progressive hearing loss, cell deterioration, dysregulation of the antioxidant defense system, and chronic inflammation, among others. To further characterize cellular and molecular mechanisms at the crossroads between noise and age-related hearing loss (ARHL), 3-month-old rats were exposed to a noise-accelerated presbycusis (NAP) protocol and tested at 6 and 16 months of age, using auditory brainstem responses, Real-Time Reverse Transcription-Quantitative PCR (RT-qPCR) and immunocytochemistry. Chronic noise-exposure leading to permanent auditory threshold shifts in 6-month-old rats, resulted in impaired sodium/potassium activity, degenerative changes in the lateral wall and spiral ganglion, increased lipid peroxidation, and sustained cochlear inflammation with advancing age. Additionally, at 6 months, noise-exposed rats showed significant increases in the gene expression of antioxidant enzymes (superoxide dismutase 1/2, glutathione peroxidase 1, and catalase) and inflammation-associated molecules [ionized calcium binding adaptor molecule 1, interleukin-1 beta (IL-1ß), and tumor necrosis factor-alpha]. The levels of IL-1ß were upregulated in the spiral ganglion and spiral ligament, particularly in type IV fibrocytes; these cells showed decreased levels of connective tissue growth factor and increased levels of 4-hydroxynonenal. These data provide functional, structural and molecular evidence that age-noise interaction contributes to exacerbating presbycusis in young rats by leading to progressive dysfunction and early degeneration of cochlear cells and structures. These findings contribute to a better understanding of NAP etiopathogenesis, which is essential as it affects the life quality of young adults worldwide.

Frailty Syndrome and Oxidative Stress as Possible Links Between Age-Related Hearing Loss and Alzheimer's Disease.

As it is well known, a worldwide improvement in life expectancy has taken place. This has brought an increase in chronic pathologies associated with aging. Cardiovascular, musculoskeletal, psychiatric, and neurodegenerative conditions are common in elderly subjects. As far as neurodegenerative diseases are concerned dementias and particularly, Alzheimer's disease (AD) occupy a central epidemiological position given their high prevalence and their profound negative impact on the quality of life and life expectancy. The amyloid cascade hypothesis partly explains the immediate cause of AD. However, limited therapeutical success based on this hypothesis suggests more complex remote mechanisms underlying its genesis and development. For instance, the strong association of AD with another irreversible neurodegenerative pathology, without curative treatment and complex etiology such as presbycusis, reaffirms the intricate nature of the etiopathogenesis of AD. Recently, oxidative stress and frailty syndrome have been proposed, independently, as key factors underlying the onset and/or development of AD and presbycusis. Therefore, the present review summarizes recent findings about the etiology of the above-mentioned neurodegenerative diseases, providing a critical view of the possible interplay among oxidative stress, frailty syndrome, AD and presbycusis, that may help to unravel the common mechanisms shared by both pathologies. This knowledge would help to design new possible therapeutic strategies that in turn, will improve the quality of life of these patients.

Axon morphologies and convergence patterns of projections from different sensory-specific cortices of the anterior ectosylvian sulcus onto multisensory neurons in the cat superior colliculus.

Corticofugal projections to the thalamus reveal 2 axonal morphologies, each associated with specific physiological attributes. These determine the functional characteristics of thalamic neurons. It is not clear, however, whether such features characterize the corticofugal projections that mediate multisensory integration in superior colliculus (SC) neurons. The cortico-collicular projections from cat anterior ectosylvian sulcus (AES) are derived from its visual, auditory, and somatosensory representations and are critical for multisensory integration. Following tracer injections into each subdivision, 2 types of cortico-collicular axons were observed. Most were categorized as type I and consisted of small-caliber axons traversing long distances without branching, bearing mainly small boutons. The less frequent type II had thicker axons, more complex branching patterns, larger boutons, and more complex terminal boutons. Following combinatorial injections of 2 different fluorescent tracers into defined AES subdivisions, fibers from each were seen converging onto individual SC neurons and indicate that such convergence, like that in the corticothalamic system, is mediated by 2 distinct morphological types of axon terminals. Nevertheless, and despite the conservation of axonal morphologies across different subcortical systems, it is not yet clear if the concomitant physiological attributes described in the thalamus are directly applicable to multisensory integration.

Antioxidants and Vasodilators for the Treatment of Noise-Induced Hearing Loss: Are They Really Effective?

We live in a world continuously immersed in noise, an environmental, recreational, and occupational factor present in almost every daily human activity. Exposure to high-level noise could affect the auditory function of individuals at any age, resulting in a condition called noise-induced hearing loss (NIHL). Given that by 2018, more than 400 million people worldwide were suffering from disabling hearing loss and that about one-third involved noise over-exposure, which represents more than 100 million people, this hearing impairment represents a serious health problem. As of today, there are no therapeutic measures available to treat NIHL. Conventional preventive measures, including public awareness and education and physical barriers to noise, do not seem to suffice, as the population is still being affected by damaging noise levels. Therefore, it is necessary to develop or test pharmacological agents that may prevent and/or diminish the impact of noise on hearing. Data availability about the pathophysiological processes involved in triggering NIHL has allowed researchers to use compounds, that could act as effective therapies, by targeting specific mechanisms such as the excess generation of free radicals and blood flow restriction to the cochlea. In this review, we summarize the advantages/disadvantages of these therapeutic agents, providing a critical view of whether they could be effective in the human clinic.

Oral Antioxidant Vitamins and Magnesium Limit Noise-Induced Hearing Loss by Promoting Sensory Hair Cell Survival: Role of Antioxidant Enzymes and Apoptosis Genes.

Noise induces oxidative stress in the cochlea followed by sensory cell death and hearing loss. The proof of principle that injections of antioxidant vitamins and Mg2+ prevent noise-induced hearing loss (NIHL) has been established. However, effectiveness of oral administration remains controversial and otoprotection mechanisms are unclear. Using auditory evoked potentials, quantitative PCR, and immunocytochemistry, we explored effects of oral administration of vitamins A, C, E, and Mg2+ (ACEMg) on auditory function and sensory cell survival following NIHL in rats. Oral ACEMg reduced auditory thresholds shifts after NIHL. Improved auditory function correlated with increased survival of sensory outer hair cells. In parallel, oral ACEMg modulated the expression timeline of antioxidant enzymes in the cochlea after NIHL. There was increased expression of glutathione peroxidase-1 and catalase at 1 and 10 days, respectively. Also, pro-apoptotic caspase-3 and Bax levels were diminished in ACEMg-treated rats, at 10 and 30 days, respectively, following noise overstimulation, whereas, at day 10 after noise exposure, the levels of anti-apoptotic Bcl-2, were significantly increased. Therefore, oral ACEMg improves auditory function by limiting sensory hair cell death in the auditory receptor following NIHL. Regulation of the expression of antioxidant enzymes and apoptosis-related proteins in cochlear structures is involved in such an otoprotective mechanism.

Cortex contacts both output neurons and nitrergic interneurons in the superior colliculus: direct and indirect routes for multisensory integration.

The ability of cat superior colliculus (SC) neurons to integrate information from different senses is thought to depend on direct projections from regions along the anterior ectosylvian sulcus (AES). However, electrical stimulation of AES also activates SC output neurons polysynaptically. In the present study, we found that nitric oxide (NO)-containing (nitrergic) interneurons are a target of AES projections, forming a component of this cortico-SC circuitry. The dendritic and axonal processes of these corticorecipient nitrergic interneurons apposed the soma and dendrites of presumptive SC output neurons. Often, an individual cortical fiber targeted both an output neuron and a neighboring nitrergic interneuron that, in turn, contacted the output neuron. Many (46%) nitrergic neurons also colocalized with gamma-aminobutyric acid (GABA), suggesting that a substantial subset have the potential for inhibiting output neurons. These observations suggest that nitrergic interneurons are positioned to convey cortical influences onto SC output neurons disynaptically via nitrergic mechanisms as well as conventional neurotransmitter systems utilizing GABA and other, possibly excitatory, neurotransmitters. In addition, because NO also acts as a retrograde messenger, cortically mediated NO release from the postsynaptic elements of nitrergic interneurons could influence presynaptic cortico-SC terminals that directly contact output neurons.

Age-Related Hearing Loss Is Accelerated by Repeated Short-Duration Loud Sound Stimulation.

Both age-related hearing loss (ARHL) and noise-induced hearing loss (NIHL) may share pathophysiological mechanisms in that they are associated with excess free radical formation and cochlear blood flow reduction, leading to cochlear damage. Therefore, it is possible that short, but repeated exposures to relatively loud noise during extended time periods, like in leisure (i.e., musical devices and concerts) or occupational noise exposures, may add to cochlear aging mechanisms, having an impact on the onset and/or progression of ARHL. Consequently, the aim of the present study was to determine if repeated short-duration overexposure to a long-term noise could accelerate permanent auditory threshold shifts associated with auditory aging in an animal model of ARHL. Toward this goal, young adult, 3-month-old Wistar rats were divided into two groups: one exposed (E) and the other non-exposed (NE) to noise overstimulation. The stimulation protocol consisted of 1 h continuous white noise at 110 dB sound pressure level (SPL), 5 days a week, allowing 2 days for threshold recovery before initiating another stimulation round, until the animals reached an age of 18 months. Auditory brainstem response (ABR) recordings at 0.5, 1, 2, 4, 8, 16, and 32 kHz were performed at 3, 6, 12, and 18 months of age. The results demonstrate that in the E group there were significant increases in auditory thresholds at all tested frequencies starting already at 6 months of age, which extended at 12 and 18 months. However, in NE animals threshold shifts were not evident until 12 months, extending to 18 months of age. Threshold shifts observed in the E animals at 6 and 12 months were significantly larger than those observed in the NE group at the same ages. Threshold shifts at 6 and 12 months in E animals resembled those at 12 and 18 months in NE animals, respectively. This suggests that repeated noise overstimulation in short-duration episodes accelerates the time-course of hearing loss in this animal model of ARHL.

Neuroglial Involvement in Abnormal Glutamate Transport in the Cochlear Nuclei of the Igf1 -/- Mouse.

Insulin-like growth factor 1 (IGF-1) is a powerful regulator of synaptic activity and a deficit in this protein has a profound impact on neurotransmission, mostly on excitatory synapses in both the developing and mature auditory system. Adult Igf1 -/- mice are animal models for the study of human syndromic deafness; they show altered cochlear projection patterns into abnormally developed auditory neurons along with impaired glutamate uptake in the cochlear nuclei, phenomena that probably reflect disruptions in neuronal circuits. To determine the cellular mechanisms that might be involved in regulating excitatory synaptic plasticity in 4-month-old Igf1 -/- mice, modifications to neuroglia, astroglial glutamate transporters (GLTs) and metabotropic glutamate receptors (mGluRs) were assessed in the cochlear nuclei. The Igf1 -/- mice show significant decreases in IBA1 (an ionized calcium-binding adapter) and glial fibrillary acidic protein (GFAP) mRNA expression and protein accumulation, as well as dampened mGluR expression in conjunction with enhanced glutamate transporter 1 (GLT1) expression. By contrast, no differences were observed in the expression of glutamate aspartate transporter (GLAST) between these Igf1 -/- mice and their heterozygous or wildtype littermates. These observations suggest that congenital IGF-1 deficiency may lead to alterations in microglia and astrocytes, an upregulation of GLT1, and the downregulation of groups I, II and III mGluRs. Understanding the molecular, biochemical and morphological mechanisms underlying neuronal plasticity in a mouse model of hearing deficits will give us insight into new therapeutic strategies that could help to maintain or even improve residual hearing when human deafness is related to IGF-1 deficiency.

Decreased levels of GABA in the inferior colliculus of the epilepsy-prone hamster (GPG/Vall).

Abnormalities in GABA levels in the central nucleus of the inferior colliculus (CNIC) of the epilepsy-prone hamster (GPG/Vall) were evaluated by using immunohistochemistry, densitometry and high performance liquid chromatography (HPLC). These findings demonstrate a decrease both in GABA immunostaining (neuropil and neurons) and in GABA concentration (HPLC) in the CNIC of the epileptic hamster compared to control animals. These decreases may reflect a reduced availability of this neurotransmitter that may act as an audiogenic seizure-initiating factor.

An Oral Combination of Vitamins A, C, E, and Mg++ Improves Auditory Thresholds in Age-Related Hearing Loss.

The increasing rate of age-related hearing loss (ARHL), with its subsequent reduction in quality of life and increase in health care costs, requires new therapeutic strategies to reduce and delay its impact. The goal of this study was to determine if ARHL could be reduced in a rat model by administering a combination of antioxidant vitamins A, C, and E acting as free radical scavengers along with Mg++, a known powerful cochlear vasodilator (ACEMg). Toward this goal, young adult, 3 month-old Wistar rats were divided into two groups: one was fed with a diet composed of regular chow ("normal diet," ND); the other received a diet based on chow enriched in ACEMg ("enhanced diet," ED). The ED feeding began 10 days before the noise stimulation. Auditory brainstem recordings (ABR) were performed at 0.5, 1, 2, 4, 8, 16, and 32 kHz at 3, 6-8, and 12-14 months of age. No differences were observed at 3 months of age, in both ND and ED animals. At 6-8 and 12-14 months of age there were significant increases in auditory thresholds and a reduction in the wave amplitudes at all frequencies tested, compatible with progressive development of ARHL. However, at 6-8 months threshold shifts in ED rats were significantly lower in low and medium frequencies, and wave amplitudes were significantly larger at all frequencies when compared to ND rats. In the oldest animals, differences in the threshold shift persisted, as well as in the amplitude of the wave II, suggesting a protective effect of ACEMg on auditory function during aging. These findings indicate that oral ACEMg may provide an effective adjuvant therapeutic intervention for the treatment of ARHL, delaying the progression of hearing impairment associated with age.

Morphologic and neurochemical alterations in the superior colliculus of the genetically epilepsy-prone hamster (GPG/Vall).

The GPG/Vall hamster is an animal model that exhibits seizures in response to sound stimulation. Since the superior colliculus (SC) is implicated in the neuronal network of audiogenic seizures (AGS) in other forms of AGS, this study evaluated seizure-related anatomical or neurochemical abnormalities in the SC of the GPG/Vall hamster. This involved calbindin (CB) and parvalbumin (PV) immunohistochemistry, densitometric analysis and high performance liquid chromatography in the superficial and deep layers of the SC in control and epileptic animals. Compared to control animals, a reduction in SC volume and a hypertrophy of neurons located in the deep layers of the SC were observed in the epileptic hamster. Although, analysis of CB-immunohistochemistry in the superficial layers did not show differences between groups, analysis of PV-immunostaining in the deep SC revealed an increase in the mean gray level within immunostained neurons as well as a decreased immunostained neuropil in the GPG/Vall hamster as compared to control animals. These alterations were accompanied by a decrease in the levels of GABA and increased levels of taurine in the epileptic animal. These data indicate that the deep SC of the GPG/Vall hamster is structurally abnormal; suggesting its involvement in the neuronal network for AGS.

The Role of Glia in the Peripheral and Central Auditory System Following Noise Overexposure: Contribution of TNF-α and IL-1ß to the Pathogenesis of Hearing Loss.

Repeated noise exposure induces inflammation and cellular adaptations in the peripheral and central auditory system resulting in pathophysiology of hearing loss. In this study, we analyzed the mechanisms by which noise-induced inflammatory-related events in the cochlea activate glial-mediated cellular responses in the cochlear nucleus (CN), the first relay station of the auditory pathway. The auditory function, glial activation, modifications in gene expression and protein levels of inflammatory mediators and ultrastructural changes in glial-neuronal interactions were assessed in rats exposed to broadband noise (0.5-32 kHz, 118 dB SPL) for 4 h/day during 4 consecutive days to induce long-lasting hearing damage. Noise-exposed rats developed a permanent threshold shift which was associated with hair cell loss and reactive glia. Noise-induced microglial activation peaked in the cochlea between 1 and 10D post-lesion; their activation in the CN was more prolonged reaching maximum levels at 30D post-exposure. RT-PCR analyses of inflammatory-related genes expression in the cochlea demonstrated significant increases in the mRNA expression levels of pro- and anti-inflammatory cytokines, inducible nitric oxide synthase, intercellular adhesion molecule and tissue inhibitor of metalloproteinase-1 at 1 and 10D post-exposure. In noise-exposed cochleae, interleukin-1ß (IL-1ß), and tumor necrosis factor α (TNF-α) were upregulated by reactive microglia, fibrocytes, and neurons at all time points examined. In the CN, however, neurons were the sole source of these cytokines. These observations suggest that noise exposure causes peripheral and central inflammatory reactions in which TNF-α and IL-1ß are implicated in regulating the initiation and progression of noise-induced hearing loss.

Noise-Induced "Toughening" Effect in Wistar Rats: Enhanced Auditory Brainstem Responses Are Related to Calretinin and Nitric Oxide Synthase Upregulation.

An appropriate conditioning noise exposure may reduce a subsequent noise-induced threshold shift. Although this "toughening" effect helps to protect the auditory system from a subsequent traumatic noise exposure, the mechanisms that regulate this protective process are not fully understood yet. Accordingly, the goal of the present study was to characterize physiological processes associated with "toughening" and to determine their relationship to metabolic changes in the cochlea and cochlear nucleus (CN). Auditory brainstem responses (ABR) were evaluated in Wistar rats before and after exposures to a sound conditioning protocol consisting of a broad-band white noise of 118 dB SPL for 1 h every 72 h, four times. After the last ABR evaluation, animals were perfused and their cochleae and brains removed and processed for the activity markers calretinin (CR) and neuronal nitric oxide synthase (nNOS). Toughening was demonstrated by a progressively faster recovery of the threshold shift, as well as wave amplitudes and latencies over time. Immunostaining revealed an increase in CR and nNOS levels in the spiral ganglion, spiral ligament, and CN in noise-conditioned rats. Overall, these results suggest that the protective mechanisms of the auditory toughening effect initiate in the cochlea and extend to the central auditory system. Such phenomenon might be in part related to an interplay between CR and nitric oxide signaling pathways, and involve an increased cytosolic calcium buffering capacity induced by the noise conditioning protocol.

Quantitative changes in calretinin immunostaining in the cochlear nuclei after unilateral cochlear removal in young ferrets.

Neurons of the cochlear nuclei receive axosomatic endings from primary afferent fibers from the cochlea and have projections that diverge to form parallel ascending auditory pathways. These cells are characterized by neurochemical phenotypes such as levels of calretinin. To test whether or not early deafferentation results in changes in calretinin immunostaining in the cochlear nucleus, unilateral cochlear ablations were performed in ferrets soon after hearing onset (postnatal day [P]30-P40). Two months later, changes in calretinin immunostaining as well as cell size, volume, and synaptophysin immunostaining were assessed in the anteroventral (AVCN), posteroventral (PVCN), and dorsal cochlear nucleus (DCN). A decrease in calretinin immunostaining was evident ipsilaterally within the AVCN and PVCN but not in the DCN. Further analysis revealed a decrease both in the calretinin-immunostained neuropil and in the calretinin-immunostained area within AVCN and PVCN neurons. These declines were accompanied by significant ipsilateral decreases in volume as well as neuron area in the AVCN and PVCN compared with the contralateral cochlear nucleus and unoperated animals, but not compared with the DCN. In addition, there was a significant contralateral increase in calretinin-immunostained area within AVCN and PVCN neurons compared with control animals. Finally, a decrease in area of synaptophysin immunostaining in both the ipsilateral AVCN and PVCN without changes in the number of boutons was found. The present data demonstrate that unilateral cochlear ablation leads to 1) decreased immunostaining of the neuropil in the AVCN and PVCN ipsilaterally, 2) decreased calretinin immunostaining within AVCN and PVCN neurons ipsilaterally, 3) synaptogenesis in the AVCN and PVCN ipsilaterally, and 4) increased calretinin immunostaining within AVCN and PVCN neurons contralaterally.

Validation of Reference Genes for RT-qPCR Analysis in Noise-Induced Hearing Loss: A Study in Wistar Rat.

The reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) requires adequate normalization in order to ensure accurate results. The use of reference genes is the most common method to normalize RT-qPCR assays; however, many studies have reported that the expression of frequently used reference genes is more variable than expected, depending on experimental conditions. Consequently, proper validation of the stability of reference genes is an essential step when performing new gene expression studies. Despite the fact that RT-qPCR has been widely used to elucidate molecular correlates of noise-induced hearing loss (NIHL), up to date there are no reports demonstrating validation of reference genes for the evaluation of changes in gene expression after NIHL. Therefore, in this study we evaluated the expression of some commonly used reference genes (Arbp, b-Act, b2m, CyA, Gapdh, Hprt1, Tbp, Tfrc and UbC) and examined their suitability as endogenous control genes for RT-qPCR analysis in the adult Wistar rat in response to NIHL. Four groups of rats were noise-exposed to generate permanent cochlear damage. Cochleae were collected at different time points after noise exposure and the expression level of candidate reference genes was evaluated by RT-qPCR using geNorm, NormFinder and BestKeeper software to determine expression stability. The three independent applications revealed Tbp as the most stably expressed reference gene. We also suggest a group of top-ranked reference genes that can be combined to obtain suitable reference gene pairs for the evaluation of the effects of noise on gene expression in the cochlea. These findings provide essential basis for further RT-qPCR analysis in studies of NIHL using Wistar rats as animal model.

Synergistic effects of free radical scavengers and cochlear vasodilators: a new otoprotective strategy for age-related hearing loss.

The growing increase in age-related hearing loss (ARHL), with its dramatic reduction in quality of life and significant increase in health care costs, is a catalyst to develop new therapeutic strategies to prevent or reduce this aging-associated condition. In this regard, there is extensive evidence that excessive free radical formation along with diminished cochlear blood flow are essential factors involved in mechanisms of other stress-related hearing loss, such as that associated with noise or ototoxic drug exposure. The emerging view is that both play key roles in ARHL pathogenesis. Therapeutic targeting of excessive free radical formation and cochlear blood flow regulation may be a useful strategy to prevent onset of ARHL. Supporting this idea, micronutrient-based therapies, in particular those combining antioxidants and vasodilators like magnesium (Mg(2+)), have proven effective in reducing the impact of noise and ototoxic drugs in the inner ear, therefore improving auditory function. In this review, the synergistic effects of combinations of antioxidant free radicals scavengers and cochlear vasodilators will be discussed as a feasible therapeutic approach for the treatment of ARHL.

Alterations in calretinin immunostaining in the ferret superior olivary complex after cochlear ablation.

In this study, we used image analysis to assess changes in calretinin immunoreactivity in the lateral (LSO) and medial (MSO) superior olivary nuclei in ferrets 2 months after unilateral cochlear ablations at 30-40 days of age, soon after hearing onset. These two nuclei are the first significant sites of binaural convergence in the ascending auditory system, and both receive direct projections from the deafferented cochlear nucleus. Cochlear ablation results in a decrease in the overall level of calretinin immunostaining within the LSO ipsilaterally compared with the contralateral side and with control animals and within the MSO bilaterally compared with control ferrets. In addition, the level of calretinin immunostaining ipsilaterally within neurons in the LSO was significantly less in cochlear ablated than control animals. In contrast, there was no effect of cochlear ablation on the level of calretinin immunostaining within neurons either in the contralateral LSO or in the MSO. These results are consistent with a downregulation in calretinin within the neuropil of MSO bilaterally and LSO ipsilaterally, as well as a downregulation in calretinin within somata in the ipsilateral LSO as a result of unilateral cochlear ablation soon after hearing onset. Thus, cochlear-driven activity appears to affect calcium binding protein levels in both neuropil and neurons within the superior olivary complex.

Upregulation of calretinin immunostaining in the ferret inferior colliculus after cochlear ablation.

In many systems, including ascending auditory pathways, calcium-binding proteins are markers of specific neuronal circuits. Previous studies suggest that calretinin immunostaining may be a specific marker for circuits in the inferior colliculus (IC) that code timing information. We undertook experiments to determine the changes in calretinin immunostaining in the IC that take place in response to cochlear ablation. Cochlear ablation was performed unilaterally in ferrets just after hearing onset. Animals survived for 2-3 months after ablation and brains were then processed for calretinin immunocytochemistry. The mean optical density and stained area of the calretinin immunopositive plexus in the IC were determined for five coronal sections through the right and left IC. In controls (n = 3), measurements of these parameters in the central nucleus of the IC showed symmetry between the two sides. In experimental animals (n = 8) the calretinin immunopositive plexus contralateral to the cochlear ablation was denser and larger than that in either the ipsilateral IC or in the IC of control animals. The calretinin plexus in the ipsilateral IC was slightly less dense and smaller than in controls but the differences did not reach statistical significance. IC volume measurements and synaptophysin immunostaining analysis in the central nucleus of the IC revealed no statistical differences between ablated and control animals or between the two sides in ablated animals. The significant increase in both mean optical density and immunostained area of the calretinin plexus in the IC contralateral to the cochlear ablation may reflect an upregulation in calretinin expression in the nuclei that contribute to this plexus.

Quantitative measurement of afferent layers in the ferret inferior colliculus: DNLL projections to sublayers.

In the central nucleus of the inferior colliculus (IC), afferent projections are aligned with dendritic arbors of disk-shaped cells, forming fibrodendritic layers. One feature that may serve as a guide for study of the intrinsic organization of the IC layers is the segregation of certain inputs to bands and patches within the layers of the central nucleus. In this study, we used Phaseolus leucoagglutinin as an anterograde tracer to examine the projections from the dorsal nucleus of the lateral lemniscus to the contralateral IC in adult ferrets. The labeled afferent projections distributed along the IC layers in a series of bands where there were dense endings and interband spaces where there were few if any endings. Branches of individual labeled axons that were reconstructed distributed within a single afferent band. Measurements of both the terminal density distribution and the optical density across the band were similar indicating that afferent bands were approximately 85 microm thick. Quantitative measurements of the labeled afferent bands will enhance comparison with other afferent projections and analysis of afferent development and plasticity.